bd logoRhapsody Sequence Analysis Pipeline N22_TCR
Pipeline Version: 2.0
Wed Jan 03 2024 14:06:20 GMT+0000 (Greenwich Mean Time)

Summary

Library
Total Reads in FASTQ
Pct Reads Passing Quality Filter
Pct CellLabel UMI Aligned Uniquely
N22 TCR 83,671,440 38.03% 39.55%
Putative Cells

85,988

Bioproduct Type
Aligned Reads By Type
Mean Reads per Cell
Mean Molecules per Cell
mRNA 3,875,539 32.62 29.79
Sample Tags 18,520 0.05 -
VDJ TCR 542,230 2.93 0.18

Graphs

No graphs available. (Usually due to too few or too many cells)

Sequence Quality

Library
Total Reads in FASTQ
Pct Read Pair Overlap
Pct Reads Too Short
Pct Reads Low Base Quality
Pct Reads High SNF
Pct Reads Filtered Out
Total Reads After Quality Filtering
N22 TCR 83,671,440 31.56% 58.09% 0.21% 3.67% 61.97% 31,817,242

Library Quality

Library
Total Filtered Reads
Pct Q30 Bases in Filtered R2
Pct CellLabel UMI
Pct CellLabel UMI Aligned Uniquely
Pct Reads Useful
N22 TCR 31,817,242 62.94% 48.18% 39.55% 13.94%

Alignment Categories

Library
CellLabel UMI Reads
Annotated Transcriptome Pct
Introns Pct
Intergenic Regions Pct
Antisense Pct
Not Unique Pct
Ambiguous Pct
No Feature Pct
AbSeq Pct
Sample Tag Pct
VDJ TCR Pct
VDJ BCR Pct
Unaligned Pct
N22 TCR 15,329,454 1.53% 23.75% 17.47% 13.22% 21.5% 0.92% 0.04% 0% 0.12% 3.54% 0% 17.92%

Reads and Molecules

Bioproduct Type
Aligned Reads By Type
Total Raw Molecules
Total RSEC Molecules
Mean Raw Sequencing Depth
Mean RSEC Sequencing Depth
Sequencing Saturation
mRNA 3,875,539 3,560,922 3,556,521 1.09 1.09 14.56

Cells

Putative Cells


Basic Algorithm Cells
Bioproduct Type
Pct Reads from Putative Cells
Mean Reads per Cell
Median Reads per Cell
Mean Molecules per Cell
Median Molecules per Cell
Mean Bioproducts per Cell
Median Bioproducts per Cell
Total Bioproducts Detected
mRNA 72.37% 32.62 27 29.79 25 18.45 16 24,220

Sample Multiplexing

Valid Reads aligned to Sample Tags

Pct Reads from Putative Cells

Sample Tag
Sample Names
Raw Reads
Pct of Raw Reads
Cells Called
Pct of Putative Cells Called
Raw Reads in Called Cells
Mean Reads per Called Cell
All_Tags - 18,520 100% 85,988 100% 3,894 0.05
SampleTag01_flex - 1,658 8.95% 1 0% 220 220
SampleTag02_flex - 3,814 20.59% 72 0.08% 890 12.36
SampleTag03_flex - 1,550 8.37% 0 0% 0 0
SampleTag04_flex - 1,652 8.92% 25 0.03% 284 11.36
SampleTag05_flex - 2,789 15.06% 49 0.06% 588 12
SampleTag06_flex - 3,841 20.74% 64 0.07% 1,306 20.41
SampleTag07_flex - 1,363 7.36% 16 0.02% 168 10.5
SampleTag08_flex - 1,848 9.98% 30 0.03% 438 14.6
SampleTag09_flex - Not_Detected - - - - -
SampleTag10_flex - Not_Detected - - - - -
SampleTag11_flex - Not_Detected - - - - -
SampleTag12_flex - 2 0.01% 0 0% 0 0
SampleTag13_flex - Not_Detected - - - - -
SampleTag14_flex - Not_Detected - - - - -
SampleTag15_flex - 1 0.01% 0 0% 0 0
SampleTag16_flex - Not_Detected - - - - -
SampleTag17_flex - Not_Detected - - - - -
SampleTag18_flex - 1 0.01% 0 0% 0 0
SampleTag19_flex - 1 0.01% 0 0% 0 0
SampleTag20_flex - Not_Detected - - - - -
SampleTag21_flex - Not_Detected - - - - -
SampleTag22_flex - Not_Detected - - - - -
SampleTag23_flex - Not_Detected - - - - -
SampleTag24_flex - Not_Detected - - - - -
Multiplet - - - 0 0% - -
Undetermined - - - 85,731 99.7% - -

VDJ

Chain Category
Reads Cellular Aligned to VDJ
Reads Contig Assembled
Reads VDJ Annotated
Reads Putative
Reads Corrected
Pct Reads Corrected
Mean Reads Corrected per Putative Cell
data:image/svg+xml;base64,PHN2ZyB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciIHdpZHRoPSIxNiIgaGVpZ2h0PSIxNiIgZmlsbD0iY3VycmVudENvbG9yIiBjbGFzcz0iYmkgYmktY2hldnJvbi1iYXItdXAiIHZpZXdCb3g9IjAgMCAxNiAxNiI+CiAgPHBhdGggZmlsbC1ydWxlPSJldmVub2RkIiBkPSJNMy42NDYgMTEuODU0YS41LjUgMCAwIDAgLjcwOCAwTDggOC4yMDdsMy42NDYgMy42NDdhLjUuNSAwIDAgMCAuNzA4LS43MDhsLTQtNGEuNS41IDAgMCAwLS43MDggMGwtNCA0YS41LjUgMCAwIDAgMCAuNzA4ek0yLjQgNS4yYzAgLjIyLjE4LjQuNC40aDEwLjRhLjQuNCAwIDAgMCAwLS44SDIuOGEuNC40IDAgMCAwLS40LjR6Ii8+Cjwvc3ZnPg== TCR 542,230 524,574 316,056 294,636 252,297 48.1% 2.93
TCR Alpha - 41,853 19,590 18,409 14,838 35.45% 0.17
TCR Beta - 461,099 289,144 269,286 231,120 50.12% 2.69
TCR Delta - 2,309 781 752 721 31.23% 0.01
TCR Gamma - 19,313 6,541 6,189 5,618 29.09% 0.07
Chain Category
Mean Molecules Corrected per Putative Cell
Molecules VDJ Annotated
Molecules Corrected
Dominant Contigs Mean Nucleotide Length
Dominant Contigs Pct Full Length
Dominant Contigs Pct With CDR3
data:image/svg+xml;base64,PHN2ZyB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciIHdpZHRoPSIxNiIgaGVpZ2h0PSIxNiIgZmlsbD0iY3VycmVudENvbG9yIiBjbGFzcz0iYmkgYmktY2hldnJvbi1iYXItdXAiIHZpZXdCb3g9IjAgMCAxNiAxNiI+CiAgPHBhdGggZmlsbC1ydWxlPSJldmVub2RkIiBkPSJNMy42NDYgMTEuODU0YS41LjUgMCAwIDAgLjcwOCAwTDggOC4yMDdsMy42NDYgMy42NDdhLjUuNSAwIDAgMCAuNzA4LS43MDhsLTQtNGEuNS41IDAgMCAwLS43MDggMGwtNCA0YS41LjUgMCAwIDAgMCAuNzA4ek0yLjQgNS4yYzAgLjIyLjE4LjQuNC40aDEwLjRhLjQuNCAwIDAgMCAwLS44SDIuOGEuNC40IDAgMCAwLS40LjR6Ii8+Cjwvc3ZnPg== TCR 0.18 23,949 15,754 300 28.46% 99.59%
TCR Alpha 0.03 3,991 2,866 347 42.46% 99.28%
TCR Beta 0.14 19,059 12,167 290 25.55% 99.77%
TCR Delta 0 259 231 309 26.03% 94.52%
TCR Gamma 0.01 640 490 299 27.34% 99.28%
Cell Type
Number cells
TCR Paired Chains Any Pct
TCR Paired Chains Full Pct
B 74 16.22% 8.11%
Dendritic 14 0% 0%
Monocyte classical 84,080 0.31% 0.12%
Monocyte nonclassical 20 0% 0%
Natural killer 403 10.92% 4.71%
T CD4 memory 411 30.9% 19.22%
T CD4 naive 761 12.22% 6.18%
T CD8 memory 154 25.97% 12.99%
T CD8 naive 51 9.8% 3.92%
T gamma delta 20 35% 5%

Pipeline Inputs

Pipeline version: 2.0

Pipeline Start Time: Wed Jan 03 2024 14:06:20 GMT+0000 (Greenwich Mean Time)

AbSeq Reference:
  • No value
Reference Files Archive:
  • RhapRef_Human_WTA_2023-02.tar.gz
Supplemental Reference:
  • No value
Targeted Reference:
  • No value
Reads:
  • N22_TCR_S1_R1_001.fastq.gz
  • N22_TCR_S1_R2_001.fastq.gz
Multiplexing Settings

Sample Tags Version: flex
Tag Names:

VDJ Settings

VDJ Species Version: humanTCR

Putative Cell Calling Settings

Enable Refined Putative Cell Calling: False
Exact Cell Count: No value
Expected Cell Count: No value
Putative Cell Call: mRNA

Exclude Intronic reads: False

Generate Bam output: True

Metric Alerts

(Under development)
  For the [N22_TCR] library, the [Pct_Read_Pair_Overlap] metric value of 31.56% is greater than 20% (Sequencing Quality section, WTA VDJ assay threshold).
   
  • May be due to short products in the library prep.
  • Look for evidence of this in QC traces and contact customer support for suggestions on additional cleanups.
  For the [N22_TCR] library, the [Pct_Reads_Too_Short] metric value of 58.09% is greater than 10% (Sequencing Quality section, WTA VDJ assay threshold).
   
  • May be due to short products in the library prep.
  • Look for evidence of this in QC traces and contact customer support for suggestions on additional cleanups.
  For the [N22_TCR] library, the [Pct_CellLabel_UMI] metric value of 48.18% is less than 70% (Library Quality section, WTA VDJ assay threshold).
   
  • May be due to poor sequencing or library quality, or incorrect reference panel.
  • Check Q30 scores and QC traces, and confirm that the appropriate transcriptome reference and complete AbSeq reference was used.
  • This can also occur if V1 and V2 beads are combined in a single library.
  For the [N22_TCR] library, the [Pct_CellLabel_UMI_Aligned_Uniquely] metric value of 39.55% is less than 40% (Library Quality section, WTA VDJ assay threshold).
   
  • May be due to poor sequencing or library quality; check QC traces and Illumina metrics to confirm sequencing was high quality.
  • Ensure that correct reference panel was used.
  • Ensure reads from long-read libraries have been trimmed before running the pipeline.
  For the [N22_TCR] library, the [Unaligned_Pct] metric value of 17.92% is greater than 10% (Alignment Categories section, WTA assay threshold).
   
  • May occur in experiments with low cell input. This can be addressed with additional cleanups for low cell input libraries (included in the protocol).
  • Can also result from low cell viability or poor sequencing or library quality.
  • This is expected if samples such as nuclei containing a high proportion of unprocessed transcripts are being run.
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